| BackgroundWith the increase of the aging population in the world,osteoporosis is increasing and has become a public health problem all over the world recently.The side effects of medicine?drug?treatment limited its clinical application though there are effects on preventing osteoporosis.As a non-drug treatment,exercise therapy prevents osteoporosis well,becoming the hot topic of research.However,the mechanism of exercise in the prevention and treatment of osteoporosis has not been fully elucidated yet.Previous studies have shown that the Wnt/?-catenin signaling pathway plays an important role in exercise prevents osteoporosis through osteoblast differentiation,bone remodeling,and then osteogenesis.But few studies have examined the effects of intensity and duration of exercise on activation of the Wnt/?-catenin signaling pathway,and their relationship with the prevention of senile osteoporosis.Therefore,the present study used the senescence-accelerated mouse strain6?SAMP6?as a model of senile osteoporosis,to examine the effects of different intensities and durations of running exercise on bone histomorphometry of SAMP6mice and evaluated whether the Wnt signaling pathway is involved in exercise-induced bone remodeling.In addition,mechanical loading is an important factor in bone remodeling,so this study also investigated different magnitudes and durations of mechanical loading?tension and compression?on osteoblast differentiation and examed whether Wnt/?-catenin signaling pathway was involved,testing the hypothesis that?exercise or mechanical loading promoting osteoblast differentiation and bone remodeling via Wnt/?-catenin signaling pathway,then increases osteogenesis and prevents osteoporosis.Methods and MaterialsPart 1:The effects of different intensities and durations of treadmill running exercise on osteogenesis and regulation of Wnt signaling pathway in SAMP6mice1.A total of 48 SAMP6 male mice with 3 months old were recruited in this study and were randomly assigned into two groups:5-week training group?n=24?and 9-week training group?n=24?.Then each group was divided into 4 sub-groups?n=6?:5-week non exercise control group?C1?,9-week non exercise control group?C2?,5-week low intensity exercise group?L1?,9-week low intensity exercise group?L2?,5-week of moderate intensity exercise group?M1?,9-week of moderate intensity exercise group?M2?,5-week high intensity exercise group?H1?,9-week high intensity exercise group?H2?.The Inbred mice SAMR1 with age-and sex-matched were used as the homology controls and randomly divided into 2 groups?R1,R2,n=6?.2.The mice in all exercise groups were assigned to 50min running at a 5°slope at a speed of 8 m/min,18 m/min of 28 m/min representing low,medium and high intensity.Exercise training was performed 6 days per week with 1 day of rest throughout the program.The mice in the C and R groups were housed under conventional conditions without exercise as controls.3.All the mice were sacrificed post 12h overnight after the whole training program,the tibias were removed.The right tibias were used to prepare undecalcified bone sections to determine bone histomorphometric parameters,including BV/TV,MS/BS,ES/BS,MAR,BFR,O.Th,mAR.The left tibias were used to prepare decalcified bone frozen sections for in situ hybridization staining to detect the levels of Wnt1,LRP5?LDL receptor-related protein?and?-catenin mRNA expression.ELISA was used to detect the serum level of ALP?Alkaline Phosphatase?,OCN?Osteocalcin?and PTH?ParathyroidHormone?.Part 2:The effects of mechanical Compression on the differentiation and Wnt/?-catenin signaling pathway in MC3T3-E1 osteoblast.1.MC3T3-E1 osteoblasts-like cells were cultured until confluence,detached with0.25%trypsin-EDTA,and were suspended at 2×107cells/ml with culture medium including 2%LMP agarose solution,then seeded in 6-wells BiopressTM compression culture plate?Bioflex?R Plate,USA?at 37°C and cooled until gelling at room temperature to cast into 3 mm deep to prepare 3D scaffold model.2.The Flexcell-5000CTM Compression System was used to apply cycling compression loading.Sinusoidal wave with the magnitude of 0.33Mpa,0.5Mpa and 1Mpa at 1Hz frequency were used in the present study and unload 3D scaffold model samples in Biopress plate were used as a control.At each time point of 4,6 and 8h,we analyzed each compression and uncompression control samples.3.After the whole mechanical loading program,the ALP activity of osteoblast was detected.Real-time PCR?Real-time fluorescence quantitative polymerase chain reaction?and Western blot were used to investigate the mRNA and protein expression of ALP,OCN,Runx2?Runt-related transcription factor2?and Osterix?osteoblast-specific transcription factors,OSX?and the key factors of Wnt signaling pathway such as Wnt1,LRP5,?-catenin,SOST?Sclerostin?and DVL2?Dishevelled2?.4.To determine whether the Wnt signaling pathway involved in the osteoblasts response to mechanical compression,DKK-1 was added before compression loading in the culture medium.Part 3:The effects of mechanical strain on the differentiation and Wnt/?-catenin signaling pathway in MC3T3-E1 osteoblast.1.MC3T3-E1 osteoblasts-like cells were cultured until confluence,detached with0.25%trypsin-EDTA,and were seed on elastic 6 well plates?BioFlex?Plate?with 1×105 cells/ml,which were specifically suitable for the mechanical strain of Flexcell-5000 tension System.2.Flexcell-5000 tension System was used to perform mechanical strain of the magnitude of 3%,6%and 12%and the duration of 2h,4h,8h with 0.5Hz frequency.Unloaded cells cultured on BioFlex?Plate were used as a control.3.After the whole mechanical loading program,osteoblast differentiation and Wnt/?-catenin signaling pathway were investigated as the same as Part 2.In addition,osteoblast apoptosis was detected by flow cytometry.Results:Part 11.The BV/TV?MS/BS?MAR?BFR?mAR were significantly higher in the medium intensity exercise groups than the control groups?C1 and C2??P<0.05?,whereas no significant differences of ES/BS and O.Th were observed between these groups?P>0.05?.Low intensity exercise group only significantly increased the level of MS/BS,MAR and mAR after 9 weeks exercise but not 5 weeks exercise?P<0.05?,and the efficacy were lower than medium intensity exercise.While the mice in high intensity exercise groups decreased almost the one histomorphometry indexes,but there was no significant difference?P>0.05?.Additional,not only low intensity exercise,but also medium intensity exercise had more efficacy of MS/BS,MAR and mAR after 9 weeks exercise than 5 weeks exercise.2.Five weeks medium intensity exercise?M1?only significantly increase?-catenin mRNA expression when compared to the control group?C1??P<0.05?,while there was no significant difference in the expression of Wnt1 and LRP5 mRNA between these two groups?P>0.05?.Nine weeks of both low and medium intensity exercise significantly increase Wnt1 and?-cateninmRNA expression in the tibia of SAMP6mice?P<0.05?,the efficacy of medium intensity was higher than low intensity.Only 9weeks medium intensity exercise?M2?significantly increased LRP5 mRNA expression when compared to control group?C2??P<0.05?,whereas 5 weeks exercise or 9 weeks low and high intensity exercise had no significant effect on LRP5 mRNA expression?P>0.05?.3.Both five and nine weeks of medium intensity exercise could significantly increase the serum levels of ALP and OCN in SAMP6 mice?P<0.05?,while there was no significant difference between low intensity exercise groups and control groups?P>0.05?;there was no significant difference in the serum level of PTH between all groups?P>0.05?.Part 21.Both 0.33Mpa and 0.5Mpa intensity of compressive loading significantly increased the ALP activity of MC3T3-E1 osteoblasts?P<0.05?,in which the efficacy of 0.5Mpa intensity was higher than that of 0.33Mpa intensity.There was no significant difference when performed 4h or 6h of 1Mpa intensity compressive loading?P>0.05?,and even decreased ALP activity when stimulated with 8h?P<0.05?.When concerned about duration dependent,6h of mechanical loading had more efficacy than 4h or 8h of both low and medium intensity loading?P<0.05?.2.The intensity of 0.5Mpa compressive loading significantly increased ALP,OCN,Runx2 and Osterix mRNA expression in osteoblasts?P<0.05?,which are the key factors differentiation of MC3T3-E1 osteoblast,and 6h of loading had more efficacy on these factors than 4h or 8h of loading.Only 6h of 0.33Mpa intensity loading significantly increased ALP mRNA expression in osteoblasts?P<0.05?,while no significant difference was observed in OCN,Runx2 and Osterix mRNA expression?P>0.05?.The intensity of 1Mpa compressive loading with 6h significantly promoted Runx2 mRNA expression in osteoblasts?P<0.05?,but had decreased OCN and Osterix mRNA expression when loading with 8h?P<0.05?.3.The intensity of 0.5Mpa compressive loading stimulates 6h could significantly increase LRP5 mRNA expression and reduced SOST mRNA expression in MC3T3-E1 osteoblast?P<0.05?.It also significantly increased the protein expression of Wnt1 and DVL2?P<0.05?and reduced phosphorylation of?-catenin?the ratio of P-?-catenin/?-catenin?level?P<0.05?.When DKK-1 was used to inhibit the binding of Wnt ligands and receptors,Compressive loading of 0.5Mpa intensity with 6h still significantly increased Wnt1 but no DVL2 protein?P<0.05?,it also slightly reduced the ratio of P-?-catenin/?-catenin though the efficacy was not as better as control groups?P<0.05?.Part 31.Mechanical strain significantly promoted the ALP activity of MC3T3-E1osteoblasts?P<0.05?,and the most efficacy was combined with 6%elongation intensity and 4h duration.2.Mechanical strain significantly increased the mRNA expression of OCN,Runx2and Osterix in MC3T3-E1 osteoblast,in which combination of 6%elongation intensity and 4h duration had the most efficacy.In addition,mechanical strain combination of 3%elongation intensity and 6h duration significantly increased the mRNA expression of OCN and Runx2?P<0.05?but not Osterix?P>0.05?.12%elongation intensity combined with 8h duration even decreased OCN mRNA expression?P<0.05?,while no significant difference in the mRNA expression of Runx2 and Osterix was observed?P>0.05?.3.Both 3%and 6%elongation intensity of mechanical strain significantly increased the mRNA and protein expression of Wnt1 and?-catenin in MC3T3-E1 osteoblast?P<0.05?,and decrease the mRNA expression of DKK-1 and the ratio of P-?-catenin/?-catenin?P<0.05?,in which 6%intensity had more efficacy than 3%and4h duration had more efficacy than 2h or 8h.While 12%elongation intensity induced a decrease of the protein expression of Wnt1 and increased the ratio of P-?-catenin/?-catenin?P<0.05?,in which 8hloading stimulation had the most negative effects on Wnt/?-catenin signaling pathway.4.6%elongation intensity combined with 4h duration of mechanical strain induced slightly decreased of apoptosis rate?including early apoptosis,late apoptosis and total apoptosis rate?when compared to the control group in MC3T3-E1 osteoblast,but the finding showed no significant difference?P>0.05?.Whereas 12%elongation intensity combined with 8h duration of mechanical strain significantly increased apoptosis in osteoblast,especially of late apoptosis and total apoptosis?P<0.05?.Conclusion1.Positive effects of treadmill running exercise on skeletal health and osteoporosis prevention were in an intensity and duration manner.Medium intensity of treadmill running exercise promotes bone formation and osteoblast differentiation,and then preventing and treating senile osteoporosis in SAMP6 mice.Medium intensity exercise had more efficacy than low or high intensity training,and 9 weeks training had more efficacy than 5 weeks training.2.Mechanical loadings promote MC3T3-E1 osteoblast differentiation in intensity,duration and loading type manners.The intensity of 0.5Mpa compressive loading combined with 6h duration had the most efficacy than any other intensities and durations in the 3D model.The intensity of 6%elongation strain loading combined with 4h duration had the most efficacy than any other intensities and durations in monolayer model.However,mechanical loading with high intensity and long duration had negative effects on osteoblast differentiation,and even induced cell apoptosis.3.Wnt/?-catenin signaling pathway plays an essential role in running exercise/mechanical loading promote osteoblast differentiation,bone formation,then prevents senile osteoporosis.In addition,Wnt/?-catenin signaling pathway may also interact with other signaling pathway and co-regulation of bone formation and osteogenesis,maintain skeleton health. |
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